Characterization of a buried neutral histidine residue in Bacillus circulans xylanase: NMR assignments, pH titration, and hydrogen exchange.

Bacillus circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic-aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the 1H, 13C, and 15N resonances and tautomeric forms of the imidazole rings were obtained from two-dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded 1H epsilon 2 of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 x 10(-5) s-1 at pH*7.04 and 30 degrees C) is retarded by > 10(6)-fold relative to an exposed histidine. The pKa of His 156 is unperturbed at approximately 6.5, as measured from the pH dependence of the 15N- and 1H-NMR spectra of BCX. In contrast, His 149 has a pKa < 2.3, existing in the neutral N epsilon 2H tautomeric state under all conditions examined. BCX unfolds at low pH and 30 degrees C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.     

Integrin binding human antibody constant domains--probing the C-terminal structural loops for grafting the RGD motif

Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL - a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.     

Tadpole-like Conformations of Huntingtin Exon 1 Are Characterized by Conformational Heterogeneity that Persists regardless of Polyglutamine Length

Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild-type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen–deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity.     

Phytochemical Characterization of Cannabis sativa L. Roots from Northeastern Brazil

This article describes the phytochemical study of Cannabis sativa roots from northeastern Brazil. The dried plant material was pulverized and subjected to exhaustive maceration with ethanol at room temperature, obtaining the crude ethanolic extract (Cs-EEBR). The volatile compounds were analyzed by gas chromatography coupled with mass spectrometry (GC/MS), which allowed to identify 22 compounds by comparing the linear retention index (LRI), the similarity index (SI) and the fragmentation pattern of the constituents with the literature. By this technique the major compounds identified were: friedelan-3-one and β-sitosterol. In addition, two fractions were obtained from Cs-EEBR by classical column chromatography and preparative thin layer chromatography. These fractions were analyzed by NMR and IR and together with the mass spectrometry data allowed to identify the compounds: epifriedelanol, friedelan-3-one, β-sitosterol and stigmasterol. The study contributed to the phytochemical knowledge of Cannabis sativa, specifically the roots, as there are few reports on the chemical constituents of this part of the plant.     

CO2 permeability of the rat erythrocyte membrane and its inhibition

We have studied the CO₂ permeability of the erythrocyte membrane of the rat using a mass spectrometric method that employs ¹⁸O-labelled CO₂. The method yields, in addition, the intraerythrocytic carbonic anhydrase activity and the membrane HCO₃⁻ permeability. For normal rat erythrocytes, we find at 37 °C a CO₂ permeability of 0.078 ± 0.015 cm/s, an intracellular carbonic anhydrase activity of 64,100, and a bicarbonate permeability of 2.1 × 10⁻³cm/s. We studied whether the rat erythrocyte membrane possesses protein CO₂ channels similar to the human red cell membrane by applying the potential CO₂ channel inhibitors pCMBS, Dibac, phloretin, and DIDS. Phloretin and DIDS were able to reduce the CO₂ permeability by up to 50%. Since these effects cannot be attributed to the lipid part of the membrane, we conclude that the rat erythrocyte membrane is equipped with protein CO₂ channels that are responsible for at least 50% of its CO₂ permeability.     

Development and validation of an experimental and theoretical method for the chiral discrimination of dinotefuran

Dinotefuran is a low-cost agrochemical considered a highly toxic product. In this sense, there is a need for its constant environmental, biological, and food control, aiming to ensure its use to humans as well as to preserve biodiversity and ecosystems. In the present work, we developed an experimental and theoretical method for dinotefuran chiral discrimination. According to the main results, the dinotefuran enantioselective separation was efficiently optimized by high-performance liquid chromatography evaluating the influence of different percentage compositions in the mobile phase to improve the resolution of the peaks in the chromatogram. The novelty of this work was the proposition of a reduced molecular model for the chiral selector amylose-Tris-(3,5-dimethylphenylcarbamate) polysaccharide that was able to adequately describe at the molecular level its interaction with the dinotefuran enantiomers. Besides, the thermodynamic and structural parameters obtained via density functional theory calculations pointed out the chiral discrimination as well as the enantiomeric elution order of the analyte studied, confirming the experimental data, thus validating our proposed method. Finally, hydrogen bonds and repulsive interactions played a key role in the discrimination between the diastereomeric complexes, and consequently, for the dinotefuran enantioselective separation.     

A SXRF method for determining the relative concentration of trace elements in plasma protein affected by cisplatin

This article presents an analytical method for the determination of the relative concentrations of trace elements in plasma protein by gel chromatography combined with SXRF (synchrotron radiation X-ray fluorescence). The fraction of plasma protein of male Kunming mice (body weight 24.2±0.3 g), treated with a cisplatin ip injection at a dose of 10 mg/kg, was obtained by the separation of a Sephadex G-50 column (buffered with ammonium acetate, pH 5.7). The SXRF experiments were performed at the Beijing Electron Positron Collider synchrotron radiation facility. The elements (Pt, S, Ca, Fe, Ni, Cu, Zn, Se, Br, and Sr) in the fraction of the plasma proteins (>22 kDa) were assayed using highly sensitive SXRF. The relative concentrations of elements were calculated by a normalization of Compton scattering intensity around 22 keV, after the normalization for the collection time of the X-ray spectrum and the counting of the ion chamber, and subtracting the contribution of the polycarbonate film for the supporting sample. The determination could prove that the element Pt in plasma was bound with macromolecular protein. Cu and S were present in the fraction of the protein in mice treated with cisplatin increase, and their ratios of treated/control were 1.66±0.06 and 1.78±0.33, respectively; Zn decreased to a ratio of 0.78±0.09. Our results are in agreement with others that cisplatin exposure leads to a marked loss of kidney copper and a moderate rise in kidney zinc. However, this article primarily describes one of the analytical methods used; it does not emphasize the results of the effect of cisplatin on trace elements in plasma protein.     

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364-3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P3 (basic amino acid), P1' (small a.a.), and P3' (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. kcat, Km, and kcat/Km of SGP for D-Dap(MeNHBz)-GFKFF*ALRK(Dnp)-D-R-D-R were 34.8 s-1, 0.065 microM, and 535 microM-1 s-1, respectively. Ki of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH2 for SGP was 1.2x10(-10) M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.     

Viral proteomics: global evaluation of viruses and their interaction with the host

Viruses constantly adapt to and modulate the host environment during replication and propagation. Both DNA and RNA viruses encode multifunctional proteins that interact with and modify host cell proteins. While viral genomes were the first complete sequences known, the corresponding proteomes are only now elucidated, with some surprising results. Even more daunting is the task to globally monitor the impact of viral infection on the proteome of the host cell and many technical hurdles must still be overcome in order to facilitate robust and reproducible measurements. Further complicating the picture is the dynamic nature of proteins, including post-translational modifications, enzymatic cleavage and activation or destruction by proteolytic events. Nevertheless, several promising studies have been published using high-throughput methods directly measuring protein abundance. Particularly, quantitative or semiquantitative mass spectrometry-based analysis of viral and cellular proteomes are now being used to characterize viruses and their host interaction. In addition, the full set of interactions between viral and host proteins, the interactome, is beginning to emerge, with often unexpected interactions that need to be carefully validated. In this review, we will discuss two major areas of viral proteomics: first, virion proteomics (such as the protein characterization of viral particles) and second, proteoviromics, including the viral protein interactomics and the quantitative analysis of host cell proteome during viral infection.     

The mechanism of the converter domain rotation in the recovery stroke of myosin motor protein

Upon ATP binding, myosin motor protein is found in two alternative conformations, prerecovery state M* and postrecovery state M**. The transition from one state to the other, known as the recovery stroke, plays a key role in the myosin functional cycle. Despite much recent research, the microscopic details of this transition remain elusive. A critical step in the recovery stroke is the rotation of the converter domain from “up” position in prerecovery state to “down” position in postrecovery state that leads to the swing of the lever arm attached to it. In this work, we demonstrate that the two rotational states of the converter domain are determined by the interactions within a small structural motif in the force‐generating region of the protein that can be accurately modeled on computers using atomic representation and explicit solvent. Our simulations show that the transition between the two states is controlled by a small helix (SH1) located next to the relay helix and relay loop. A small translation in the position of SH1 away from the relay helix is seen to trigger the transition from “up” state to “down” state. The transition is driven by a cluster of hydrophobic residues I687, F487, and F506 that make significant contributions to the stability of both states. The proposed mechanism agrees well with the available structural and mutational studies. Proteins 2012; © 2012 Wiley Periodicals, Inc.